The ultimate expression of the genetic makeup of a cell resides in its proteins. When and how much of a protein will be synthesized depends on RNA molecules, which not only direct the process, but also constitute the machinery which assemble the proteins from amino acids. Thus, understanding RNA metabolism is a prerequisite for elucidating gene expression in normal cells and for clarifying what has gone wrong under a variety of pathological conditions, many of which are manifested by abnormal gene expression. While considerable information has accumulated about the ribonucleases (RNases) essential to RNA metabolism, much remains to be learned about their complete number, about their physiological roles and mechanism of action, and whether and how they might be regulated. It is the long term goal of this project to define the enzymes, pathways and regulatory factors that constitute the totality of RNA metabolism in a single cell. As RNA metabolism is remarkably similar in all cells, our studies will be carried out with the bacterium, Escherichia coli, for which a large amount of information is already available. Our specific aims are: 1) Analysis of quality control of defective stable RNAs and ribonucleoproteins; 2) Examination of RNases and RNA degradation under stress conditions; 3) Determination of functional domains and mechanism of action of RNase R; 4) Elucidation of the physiological role of exoribonucleases; and 5) Identification of RNases required for maturation of ribosomal RNA. To accomplish these aims we will use biochemical, molecular biological, genetic and physiological approaches. The mutant strains and purified enzymes required to carry out this work already are available in the laboratory. We anticipate that the studies detailed in this proposal will provide important, new information on a variety of aspects of the enzymes, factors and processes that encompass RNA metabolism. Knowledge of all facets of RNA metabolism is essential if we are to understand the details of gene expression in normal cells, of what may go wrong in various disease states, and if we are to be successful in correcting pathological processes caused by altered gene expression.